Oligodeoxyribonucleoside phosphorothioates (PS-ODNs) have been, and are still, extensively studied as potential therapeutic agents against various types of cancer and infectious diseases in humans. Given that these oligonucleotide analogues are P-chiral, each of the internucleotidic phosphorothioate linkages adopts either a Rp or a Sp configuration. Stereopure Rp-(PS-ODNs) have been prepared enzymatically and exhibited a lower stability to nucleases endogenous to human serum than the parent PS-ODNs with undefined P-chirality. Conversely, chemically synthesized stereopure Sp-(PS-ODNs) have demonstrated superior stability to these nucleases than P-diastereomeric PS-ODNs. Thus, to further investigate the biological, pharmacokinetic, and toxicologic properties of P-stereopure PS-ODNs improved chemical methods are required to synthesize these biomolecules and increase their availability for clinical studies. We have observed that deoxyribonucleoside cyclic N-acylphosphoramidites are efficient monomers for the stereospecific synthesis of PS-ODNs. Indeed, base-assisted condensation of the 5'-OH function of a nucleoside or a nucleotide covalently linked to a solid support with deoxyribonucleoside cyclic N-acylphosphoramidites led to rapid and efficient formation of an internucleoside phosphite triester linkage. The phosphite triester function can be either oxidized to a phosphate triester (PO) or sulfurized to a P-stereodefined thiophosphate phosphotriester (PS) function to permit, for example, stereocontrolled synthesis of chimeric PO/PS-ODNs. A successful assignment of the absolute configuration of a deoxyribonucleoside cyclic N-acylphosphoramidite at phosphorus has been accomplished for the first time by M-GOESY nuclear magnetic resonance spectroscopy through accurate measurements of internuclear distances between diagnostic nuclei. The method is convenient, rapid, and unequivocally confirms that the condensation of deoxyribonucleoside cyclic N-acylphosphoramidites with base-activated nucleosidic 5'-hydroxyls proceeded via a single SN2 nucleophilic substitution event. The use of new deoxyribonucleoside cyclic N-acylphosphoramidite monomers and condensation conditions has enabled incorporation of the four different nucleobases into a 20-mer DNA oligonucleotide. It is important to note that three of these nucleobases were N-unprotected, thereby underscoring the versatility of cyclic phosphoramidite chemistry. Optimization of the chemistry in terms of the type of base and support being used for improving coupling yields is ongoing in an effort to apply the chemistry to the synthesis of oligonucleotides on microarrays. When investigating cylic N-acyl phosphoramidites, we have discovered that phosphate/thiophosphate protection is heat-sensitive. This discovery led to the development of a number of novel thermolabile phosphate protecting groups including the 2-(N-formyl-N-methyl)aminoethyl, 4-oxopentyl, 3-[(N-tert-butyl)carboxamido]-1-propyl, 3-(2-pyridyl)-1-propyl, and 2-[N-methyl-N-(2-pyridyl)]aminoethyl groups. Since these phosphate/thiophosphate protecting groups do not lead to DNA alkylation or thiophosphate desulfurization under the conditions used for oligonucleotide deprotection, these groups may find wide application in large-scale preparations of therapeutic oligonucleotides. We have recently found that thermolabile groups could also be adapted to the 5'-OH protection of nucleoside and oligonucleotides. Specifically, we have prepared thermolabile carbonates on the basis of our success with thermolabile phosphate protecting groups. Conceptually, these groups undergo cyclodecarboxylation when heated near 90?C in aqueous solvents to release carbon dioxide and a nucleosidic/nucleotidic 5'-OH. Several nucleoside 5'-O-carbonates have been synthesized and deprotection studies have been undertaken. The time required for complete deprotection depends on the nature of the carbonates, and ranges between 10 min to several hours. Most of these groups are completely stable in organic solvents at ambient temperature. Since two 5'-O-carbonates are not completely stable at ambient temperature, we have developed a "chemical switch" for the 5'-hydroxyl protection of deoxyribonucleosides consisting of a stable 5'-O-carbonate, which can be rapidly transformed to a thermolytically unstable carbonate when needed. This approach is quite appealing for the synthesis of DNA oligonucleotides on microarrays. We have also observed that modified sulfite derivatives have potential as 5'-/3'-OH protecting groups for nucleosides and nucleotides. These protecting groups are relatively easy to prepare and are stable to non-nucleophilic basic conditions. A modified 5'-O-sulfite protecting group is removed within 5 min during the iodine oxidation step of a solid-phase DNA synthesis cycle. Modified sulfite protecting groups can also be easily functionalized with fluorescent reporter groups for sensitive determination of coupling yields. The suitability of 5'-O-sulfite protecting groups for solid-phase oligonucleotide synthesis on CPG has recently been demonstrated through the efficient synthesis of a 20-mer. These emerging protecting groups should be valuable in the synthesis of oligonucleotides on microarrays considering the mildness of the conditions used for the cleavage of these groups. These findings along with the fact that deoxyribonucleoside cyclic N-acylphosphoramidites can, unlike conventional deoxyribonucleoside phosphoramidites, effectively produce oligonucleotides under relatively wet conditions are attractive features for parallel synthesis of oligonucleotides on arrayable surfaces. Oligonucleotide microarrays can be invaluable for analyzing gene expression from, for example, tumorigenic versus non-tumorigenic human cell lines, and high-throughput screening of point mutations and single nucleotide polymorphisms in genomic DNA that predispose human to diseases.